Recombinant DNA technology

Genetic Engineering - the insertion of DNA from one organism into

another

Recombinant DNA- made by connecting or recombining

fragments of DNA from different sources

Transgenic organism- organisms that contain functional

recomb. DNA

Mice can be made to produce Green Fluorescent Protein- GLOW

How to: 1. isolate foreign DNA

2. clone DNA into a "vehicle"

3. transfer recomb DNA into host

How to Clone a gene:

Restriction Enzymes- bacterial proteins that cleave DNA at specific sequences

usually at palindromic sequences (read same

backwards and forwards)

Step 1: Cut desired/cloned DNA with restriction enzymes

Vector: a DNA "vehicle" use to carry DNA

Mechanical

1. microinjectors

2. gene guns

Biological

1. Viral DNA

2. Plasmid - circular bacterial DNA

has a selectable marker- usually a gene that encodes

antibiotic resistance

Step 2: Splice gene: Ligate (attach) cloned DNA into vector

Step 3: Transfer recomb. DNA into new host to make transgenic org.

Clone- genetically identical copy

Sequencing of DNA

nucleotide sequence can be determined biochemically

requires many copies of the gene to be sequenced

Ingredients

radioactive dideoxynucleotides

DNA polymerases

DNA to be sequenced

New dna is synthesized, but ends when when ddNTP is incorporated

Then, DNA is separated by gel electrophoresis

Polymerase Chain Reaction

Used to amplify DNA

Can make millions of copies from 1 piece of DNA

Ingredients: Template DNA

dNTPs

DNA polymerase

Applications of DNA Technology: Uses of recomb. bacteria

Industry- bacteria have been engineered to break down pollutants

Medicine- can be used to produce hormones, other proteins

such as insulin, human growth hormone

Research- bacteria can produce proteins so that those proteins can be researched and characterized

Agriculture- bacteria engineered to fix nitrogen more efficiently introduced into plants

Transgenic Plants: pest resistant, produce human proteins

Transgenic Animals: study disease, role genes play in disease

Drosophila melanogaster

Caenorhabditis elegans

Mice

The Human Genome Project

Using DNA sequencing and other techniques to find the sequence of the genome and the location of all the

genes on the chromosomes

~80 000 genes on 46 chromosomes

Linkage Map- genetic map that shows the location of genes on the chromosomes

Frequency of crossing over is used to determine how

close genes are to each other

Applications:

Diagnosis of Human Disorders- once the location and proper sequence of a gene is known, that information can be used to screen for diseases

Cells are obtained from fetus, and propagated in cell culture to

get many copies of the genetic material

Gene Therapy: introducing a functional gene into a diseased

individual to replace the faulty copy

SCID (severe combined immunodifficiency disease)

DNA Fingerprinting: used to compare DNA from a known sample to a DNA sample from a crime suspect (or potential father)